This invention is directed to modified oligonucleotides that include one or more 2xe2x80x2-5xe2x80x2 internucleotide linkages and a modified nucleotide at one of the two nucleotides that are linked by the 2xe2x80x2,5xe2x80x2 linkage. That nucleotide is modified, for example, by incorporating a substituent at its 3xe2x80x2-position. The modified oligonucleotides of the present invention exhibit improved properties of nuclease resistance and binding affinity, and are of use as antisense oligonucleotides.
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Classical therapeutic modes have generally focused on interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. However, recently, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, maximum therapeutic effect and minimal side effects may be realized. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides. Oligonucleotides are now accepted as therapeutic agents with great promise. Oligonucleotides are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another. The concept of inhibiting gene expression through the use of sequence-specific binding of oligonucleotides to target RNA sequences, also known as antisense inhibition, has been demonstrated in a variety of systems, including living cells (for example see: Wagner et al., Science (1993) 260: 1510-1513; Milligan et al., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem. Reviews, (1990) 90:543-584; Stein et al., Cancer Res., (1988) 48:2659-2668).
The events that provide the disruption of the nucleic acid function by antisense oligonucleotides (Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts""O, P.O.P. (1987) Anti-Cancer Drug Design, 2:117-128, and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Oligonucleotides may also bind to duplex nucleic acids to form triplex complexes in a sequence specific manner via Hoogsteen base pairing (Beal et al., Science, (1991) 251:1360-1363; Young et al., Proc. Natl. Acad. Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeutic strategies are directed towards nucleic acid sequences that are involved in or responsible for establishing or maintaining disease conditions. Such target nucleic acid sequences may be found in the genomes of pathogenic organisms including bacteria, yeasts, fungi, protozoa, parasites, viruses, or may be endogenous in nature. By hybridizing to and modifying the expression of a gene important for the establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides may also be of therapeutic value when they bind to non-nucleic acid biomolecules such as intracellular or extracellular polypeptides, proteins, or enzymes. Such oligonucleotides are often referred to as xe2x80x98aptamersxe2x80x99 and they typically bind to and interfere with the function of protein targets (Griffin, et al., Blood, (1993), 81:3271-3276; Bock, et al., Nature, (1992) 355: 564-566).
Oligonucleotides and their analogs have been developed and used for diagnostic purposes, therapeutic applications and as research reagents. For use as therapeutics, oligonucleotides must be transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These critical functions depend on the initial stability of the oligonucleotides toward nuclease degradation. A serious deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is the enzymatic degradation of administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes referred to as nucleases. For oligonucleotides to be useful as therapeutics or diagnostics, the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation. For a non-cellular use such as a research reagent, oligonucleotides need not necessarily possess nuclease stability.
A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation.
Modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and the use of modified sugar moieties such as 2xe2x80x2-O-alkyl ribose. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. A number of modifications that dramatically alter the nature of the internucleotide linkage have also been reported in the literature. These include non-phosphorus linkages, peptide nucleic acids (PNA""s) and 2xe2x80x2-5xe2x80x2 linkages. Another modification to oligonucleotides, usually for diagnostic and research applications, is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
A variety of modified phosphorus-containing linkages have been studied as replacements for the natural, readily cleaved phosphodiester linkage in oligonucleotides. In general, most of them, such as the phosphorothioate, phosphoramidates, phosphonates and phosphorodithioates all result in oligonucleotides with reduced binding to complementary targets and decreased hybrid stability. In order to make effective therapeutics therefore this binding and hybrid stability of antisense oligonucleotides needs to be improved.
In an effort to study the influence of structural modifications of oligonucleotides on the stability of their duplexes with target RNA, a series of oligonucleotides containing more than 200 different modifications were synthesized and assessed for their hybridization affinity and Tm (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443). Sugar modifications studied included substitutions on the 2xe2x80x2-position of the sugar, 3xe2x80x2-b-substitution, replacement of the 4xe2x80x2-oxygen, the use of bicyclic sugars, and four member ring replacements. Several nucleobase modifications were also studied including substitutions at the 5, or 6 position of thymine, modifications of pyrimidine heterocycle and modifications of the purine heterocycle. Numerous backbone modifications were also investigated including backbones bearing phosphorus and those that did not bear a phosphorus atom, and backbones that were neutral. Based on the study of this large set of modified oligonucleotides four general approaches that may be used to improve hybridization of oligonucleotides to RNA targets were identified. These include: preorganization of the sugars and phosphates of the oligodeoxynucleotide strand into conformations favorable for hybrid formation, improved stacking of nucleobases as observed when polarizable groups are added to the heterocycle enhances binding affinity, increasing the number of H-bonds available for A-U pairing also helps binding, and neutralization of backbone charge will also facilitate the biding interactions by removing undesirable repulsive interactions (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443).
Sugars in DNA:RNA hybrid duplexes frequently adopt a C3xe2x80x2 endo conformation. Thus modifications that shift the conformational equilibrium of the sugar moieties in the single strand toward this conformation should preorganize the antisense strand for binding to RNA. Of the several sugar modifications that have been reported and studied in the literature, the incorporation of electronegative substituents such as 2xe2x80x2-fluoro or 2xe2x80x2-alkoxy shift the sugar conformation towards the northern pucker conformation. This pucker conformation further assisted in increasing the Tm of the oligonucleotide with its target. Large substituents at the 2xe2x80x2-position are, however, not well tolerated. A clear correlation between substituent size at the 2xe2x80x2-position and duplex stability has been observed and reported in the literature. Incorporation of alkyl substituents at the 2xe2x80x2-position typically leads to a significant decrease in binding affinity.
2xe2x80x2-O-alkyl substituents provide a strong positive influence on the binding affinity of oligonucleotides (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443). Small alkoxy groups were very favorable, and larger alkoxy groups at the 2xe2x80x2-position were found to be unfavorable. However, if the 2xe2x80x2-substituent contained an ethylene glycol motif, then a strong improvement in binding affinity to the target RNA was observed. This is suggested to arise from gauche interactions between the oxygen g to the 2xe2x80x2-oxygen atom results in a configuration of the side chain that is favorable for duplex formation.
It has been reported that while there are a number of stabilizing modifications to choose from when designing oligonucleotides, the combination of stabilizing features provides the best approach to improving binding affinities. It appears that modified oligonucleotides with very high RNA binding affinity need to be constructed by the combination of two or more different types of modifications, each of which contributes favorably to one of the four general factors important for binding affinity (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443).
One type of nucleic acid modification that has seen considerable interest is the 2xe2x80x2,5xe2x80x2-oligonucleotides. A number of research groups have revealed the synthesis and study of 2xe2x80x2,5xe2x80x2-oligonucleotides and nucleic acids. This modification entails the synthesis of oligonucleotides where the internucleotide linkages are not between the 3xe2x80x2 and 5xe2x80x2-positions of the sugars as in natural nucleic acids, but are between the 2xe2x80x2 and 5xe2x80x2 positions of the sugars of the nucleotide components. The 2xe2x80x2,5xe2x80x2 internucleotide linkage, and such oligonucleotides, has been under investigation from several different aspects.
2xe2x80x2,5/-Oligoadenylates (also referred to as 2-5A) are naturally occurring RNA isomers that are implicated in the regulation of cell growth and in the antiviral mechanism of interferon. Because of the poor uptake of such oligonucleotides and the relatively nonspecific endonucleolytic action of its target RNase L, chimeric oligonucleotides that incorporate 2-5A motifs together with an antisense construct for a specific target have also been studied (Lesiak et al, Bioconjugate Chem., 1993, 4, 467-472). These chimeric oligonucleotides bearing 3xe2x80x2-hydroxy groups at the 2xe2x80x2,5xe2x80x2-linkages were found to hybridize to complementary RNA and to activate the 2-5A dependent RNase. 2xe2x80x2,5/-Oligoadenylates that bear a fluoro substituent at the 3xe2x80x2-position have been synthesized, via phosphoramidite chemistry, to study the importance and role of the 3xe2x80x2-hydroxy group in the activity of such 2-5A oligonucleotides (Kovacs et al., Nucleosides and Nucleotides, 1995, 14, 1259-1267).
2xe2x80x2,5/-Oligonucleotides have also been the focus of research aimed at understanding the evolutionary bias towards 3xe2x80x2,5xe2x80x2 instead of 2xe2x80x2,5xe2x80x2 linked double helices to encode genetic information (Prakash et al, Angew. Chemie, 1997, 36, 1522-23); 2xe2x80x2,5xe2x80x2 linkages were found to be more susceptible to hydrolysis that their 3xe2x80x2,5xe2x80x2 analogs. 2xe2x80x2,5xe2x80x2 phosphodiester linked oligoribonucleotides have also been studied for their binding to RNA and DNA (Giannaris and Damha, Nucl. Acids Res., 1993, 21, 4742-4749). It was found that 2xe2x80x2,5xe2x80x2 oligoribonucleotides exhibited remarkable selectivity for complementary single stranded RNA over DNA. Further when a 3xe2x80x2,5xe2x80x2 phosphodiester deoxyribooligonucleotide was altered via replacement of some linkages with 2xe2x80x2,5xe2x80x2 phosphodiester ribonucleotide linkages, the resulting chimeric oligonucleotide exhibited a lower Tm when binding to both DNA and RNA targets (Giannaris and Damha, Nucl. Acids Res., 1993, 21, 4742-4749, Kandimalla et al, Nucl. Acids Res., 1997, 25, 370-378). This destablization of binding was also found to be related to the number of 2xe2x80x2,5xe2x80x2 linkages incorporated into the oligonucleotide. Similar mixed backbone oligonucleotide phosphorothioates have also been reported and these show greater destabilization of binding to DNA compared to RNA targets.
During studies on the 2-5A system, 3xe2x80x2-amino analogs of 2-5A trimer oligonucleotide phosphodiesters were synthesized and reported in the literature as exhibiting improved enzymatic stability towards phosphodiesterase (Pfleidereret al., Bioorg. Med. Chem. Lett., 1994, 4, 1047-1052). These short oligonucleotides were reported to have antiviral effects against HIV in peripheral blood mononuclear cells. Similar 2xe2x80x2,5xe2x80x2 oligoadenylate analogs have also been synthesized via phosphotriester chemistry and reported to be useful for suppressing the division of T-helper and T-killer cells and for treating autoimmune diseases and the host-vs.-graft response during organ transplant rejection (Tkachuk et al., U.S. Pat. No. 5,571,799, issued Nov. 5, 1996). Several patents also describe 2xe2x80x2,5xe2x80x2-oligoadenylate phosphodiesters and phosphorothioates as dual action antiviral agents capable of not only activating the latent RNase L but also inhibiting viral DNA polymerases. These oligonucleotides typically bear no substituent (i.e. are deoxy) or a hydroxyl or amino substituent on the 3xe2x80x2-position of the nucleosides participating in the 2xe2x80x2,5xe2x80x2 linkage. Conjugates with a variety of molecules and encapsulated formulations have also been described for these 2xe2x80x2,5xe2x80x2 oligonucleotides (Suhadolnik and Pfleiderer, U.S. Pat. No. 5,188,897, issued Feb. 23, 1993; Suhadolnik and Pfleiderer, U.S. Pat. No. 5,405,939, issued Apr. 11, 1995; Suhadolnik and Pfleiderer, U.S. Pat. No. 5,550,111, issued Aug. 27, 1996).
A number of groups have studied 2xe2x80x2,5xe2x80x2-3xe2x80x2-deoxyoligonucleotides. Giannaris and Damha showed that 2xe2x80x2,5xe2x80x2-oligoribonucleotides show weaker binding to complementary RNA targets compared to complementary DNA targets (Giannaris and Damha, Nucl. Acids Res., 1993, 21, 4742-4749). Alul and Hoke have reported that 2xe2x80x2,5xe2x80x2-oligo-3xe2x80x2-deoxynucleotides exhibit binding similar to that of corresponding 3xe2x80x2,5xe2x80x2-oligodeoxynucleotides when studied with a complementary RNA. However, in contrast to most antisense oligodeoxynucleotide analogs, 2xe2x80x2,5xe2x80x2 oligonucleotides do not bind to complementary DNA. It has also been reported that this change in bond connectivity from 3xe2x80x2,5xe2x80x2 to 2xe2x80x2,5xe2x80x2 confers improved resistance to nucleolytic degradation (Alul and Hoke, Antisense Res. and Dev., 1995, 5, 3-11). 2xe2x80x2,5/-Oligonucleotide analogs bearing hydroxyl, alkyl, aryl, alkoxy, alkyoxy, aryloxy, and azido substituents at the 3xe2x80x2-position have been claimed in a patent describing methods and compositions for sequence-specific hybridization of RNA (Alul, U.S. Pat. No. 5,532,130, issued Jul. 2, 1996). These oligonucleotides are reported to exhibit greater exo- and endo-nuclease resistance while hybridizing selectively to RNA and not DNA. Chimeric, 21-mer 2xe2x80x2,5xe2x80x2-oligo-3xe2x80x2-deoxynucleotide phosphorothioates that contain a short cassette of seven 3xe2x80x2,5xe2x80x2 linkages in the middle have been reported to be inhibitors of the steroid 5a-reductase in cell culture (Bohn, Hoke and Alul, Proceeding of the Twelfth International Round Table, 1996, in Nucleosides and Nucleotides, 1997, 16, 1195-1179. These chimeric phosphorothioate oligonucleotides bearing both 2xe2x80x2,5xe2x80x2 and 3xe2x80x2,5xe2x80x2 linkages exhibited potency similar to the completely 3xe2x80x2,5xe2x80x2 analogs but with significantly lower non-sequence specific effects.
2xe2x80x2,5/-Oligo-3xe2x80x2-deoxynucleotides have also been studied for their binding to RNA and DNA targets and their involvement in prebiotic development and genetic selection. Breslow""s group has synthesized 2xe2x80x2,5xe2x80x2-linked DNA and studied its binding properties to both RNA and DNA. Selective binding of RNA, but not DNA was observed (Dougherty et al., J. Am. Chem. Soc., 1992, 112, 6254-6255; Sheppard and Breslow, J. Am. Chem. Soc., 1992, 118, 9810-9811). This has been further elaborated to reveal that the duplex formed by 2xe2x80x2,5xe2x80x2-DNA with RNA has a stability (Tm) similar to that for the 3xe2x80x2,5xe2x80x2-DNA-RNA duplex but that this binding is of a hybrid nature (Prakash et al., Chem. Commun., 1996, 1793-94).
As part of the scientific study of oligonucleotides and antisense oligonucleotides, numerous modifications, besides 2xe2x80x2,5xe2x80x2 linkages, have been made to the internucleotide linkage. While many of these retain the phosphorus atom present in the natural phosphodiester linkage a number of non-phosphorus linkages have also been studied. One example of these is an unsaturated linkage of four atom chains connecting the two sugar residues of adjacent nucleotide units. Oligonucleotides bearing internucleotide linkages of the type 2xe2x80x2/3xe2x80x2-Sxe2x80x94CH2xe2x80x94CHxe2x95x905xe2x80x2CH2 and 2xe2x80x2/3xe2x80x2-Oxe2x80x94CH2xe2x80x94CHxe2x95x905xe2x80x2CH2 have been reported by Matteucci and Cao (U.S. Pat. No. 5,434,257, issued Jul. 18, 1995). These 2xe2x80x2,5xe2x80x2 linked oligonucleotides were reported to be stable in vivo, resistant to endogenous nucleases and capable of hybridizing to target nucleic acids.
Of the large number of modifications made and studied, few have progressed far enough through discovery and development to deserve clinical evaluation. Reasons underlying this include difficulty of synthesis, poor binding to target nucleic acids, lack of specificity for the target nucleic acid, poor in vitro and in vivo stability to nucleases, and poor pharmacokinetics. Several phosphorothioate oligonucleotides and derivatives are presently being used as antisense agents in human clinical trials for the treatment of various disease states. Although some improvements in diagnostic and therapeutic uses have been realized with these oligonucleotide modifications, there exists an ongoing demand for improved oligonucleotides that are easy to synthesize, are nuclease resistant and have good binding properties.
The present invention provides modified oligonucleotides that are easy to synthesize and exhibit good properties of nuclease resistance and hybridization to target nucleic acids. This and other objects of the invention will be apparent from a consideration of the specification as a whole.
The present invention provides oligonucleotides comprising a plurality of nucleotides linked together by internucleotide linkages. Each nucleotide includes a sugar portion and a base portion, and at least one of the internucleotide linkages is a 2xe2x80x2,5xe2x80x2-linkage wherein at least one of the linked nucleotides bears a 3xe2x80x2-substituent of the formula:
Zxe2x80x94R22xe2x80x94(R23)v
where:
Z is O, S, NH, or Nxe2x80x94R22xe2x80x94(R23)v 
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is R24 when Z is O;
R23 is hydrogen or R24 when Z is S, NH, or Nxe2x80x94R22xe2x80x94(R23)v;
R24 is amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, hydroxyalkyamino, hydroxydialkylamino, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, poly-ether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
v is from 0 to about 10.
The present invention further provides oligonucleotides bearing at least one 2xe2x80x2,5xe2x80x2 internucleotide linkage wherein at least one of the linked nucleotides includes an alkoxyalkoxy, dialkoxyalkoxy, hydroxyalkoxy, dihydroxyalkoxy, aminoalkoxy, alkylaminoalkoxy, dialkylaminoalkoxy, dialkylaminooxyalkoxy, haloalkoxy, dihaloalkoxy or trihaloalkoxy 3xe2x80x2-substituent and protected versions of the same.
In a preferred embodiment, the present invention provides oligonucleotides bearing at the 3xe2x80x2-position a substituent selected from the group consisting of, but not limited to, methoxyethoxy, hydroxyethoxy, dimethylaminooxyethoxy, trifluoromethylethoxy, aminopropoxy, and protected versions of the same.
The present invention also provides oligonucleotides bearing methoxyethoxy substituents at one or more 2xe2x80x2-positions on the sugar portion of the nucleotides.
The present invention further provided oligonucleotide having at least one 2xe2x80x2,5xe2x80x2 internucleotide linkage wherein at least one of the linked nucleotides includes a 3xe2x80x2-substituent having one of the formulas: 
where:
y1 is 0 or 1;
y2 is 0 to 10;
y3 is 1 to 10;
E is N(R41)(R42) or Nxe2x95x90C(R41)(R42); and
each R41 and each R42 is independently H, C1-C10 alkyl, a nitrogen protecting group, or R41 and R42 taken together form a nitrogen protecting group; or R41 and R42 taken together with the N or C atom to which they are attached form a ring structure that can include at least one heteroatom selected from N and O.
The present invention further provides oligonucleotides comprising a plurality of nucleotides linked together by internucleotide linkages, wherein the linkage is selected from a group consisting of, but not limited to, phosphorus-containing and non-phosphorus-containing linkages. Phosphorus-containing linkages include, but are not limited to, phosphodiester, phosphorothioate, phosphoramidate, alkylphosphonate, N3xe2x80x2- greater than P5xe2x80x2 phosphoramidate, phosphinate, phosphate, thiophosphate and phosphorodithioate linkages. Non-phosphorus-containing linkages include, but are not limited to, glycol, ether, all carbon atom, urea, carbamate, amide, cyclic, amine, hydroxylamine, hydrazino, -substituted amide 3, and methylene(methylimino) linkages.
In a preferred embodiment, the internucleotide linkages present in the oligonucleotides of the present invention are either all phosphodiester or all phosphorothioate. In a further preferred embodiment, the internucleotide linkages present in the oligonucleotides of the present invention are any combination of at least one phosphodiester and at least one phosphorothioate linkage.